ADVANCED MATERIALS HANDLING | APPLICATION NOTE

Protein Formulation Screening and USP <787> TestingAccuSizer ® automated particle count analysis for protein formulation optimization to minimize aggregation and perform USP <787> subvisible particulate matter testing.

INTRODUCTION—Dynamic light scattering (DLS) is a standard tool for determining

particle size of proteins, but this method does not provide

concentration information. The AccuSizer® single particle optical

sizing (SPOS) system is an ideal technique for determining

aggregated protein size and concentration in particles/mL for

sizes greater than 150 nm. Figure 1 shows Nicomp® DLS results

before and after heat stressing a monoclonal antibody. The after

stress result in blue shows the high resolution multimodal result

including larger aggregate peaks beginning at ~150 nm. The

AccuSizer cannot detect down to the 10 nm monomer size,

but but can provide quantitative data indicating the degree

of aggregation >150 nm. In this study the AccuSizer FX Nano

was used to investigate how various formulation stabilization

chemistries affected the resultant state of aggregation for a NIST

standard protein. The same AccuSizer particle counter can also be

used for USP <787> subvisible particulate matter testing.1

MATERIALS—The protein used was NIST reference material 8671, Lot 14HB-D-

002, NIST mAb, Humanized IgG1k monoclonal antibody2.

The primary size of the protein was measured on the Nicomp

dynamic light scattering (DLS) system using a 35 mW laser diode

source, APD detector. The protein aggregates were measured

on the AccuSizer FX Nano SIS system equipped with two sensors:

LE-400; 0.5 – 400 μm and FX Nano; 0.15 – 10 μm. The SIS

sampler provides accurate sample volume down to 100 μL with

sample recovery.

iFormulate3 (Figure 2) plates preloaded with formulations, designed

by DoE for rational formulation development, were used to test

aggregation levels as a function of formulation chemistry. These

evaluate four major variables of protein formulation; pH, ionic

strength, buffer, and stabilizer concentration using multivariate

experimental design.

Figure 2. iFormulate plate

The iFormulate plates used tested the following variables:

• pH: 5 – 7.6

• Ionic strength: 0 – 200 mM NaCl

• trehalose stabilizer: 0 – 10 %

• Buffer concentration: 10 – 50 mM

Figure 1. Nicomp DLS results before and after heat stress

2

METHODOLOGY—The NIST mAb was thawed and diluted to 1 mg/mL.

First 160 μL of filtered DI water was added to each well

in the iFormulate plate. Next 40 μL of NIST mAb was

added to each well. The plate was placed in an oven at

60°C (just below the protein melting point) for 24 hours.

Each well was analyzed using the AccuSizer particle

counter. The particle count data for >0.15 μm (total) and

>0.53 μm was submitted to iFormulate, who then

performed the data analysis and stability modeling.

RESULTS—The data was processed using a Pareto analysis

approach, a formal technique useful where many

possible courses of action are competing for attention.

The Pareto analysis gives ranking of variables and

interactions between variables. Figures 3 and 4 show

the Pareto analysis for >0.15 μm and >0.53 μm,

respectively. The colors indicate the relative importance

of the formulation variable to increased particle

count with blue = significant, gray = borderline, and

red = less significant.

Pareto E�ects for Total

+

Ter

m

1211109876543210

ECHIP

E�ect/1000000

+

+

++

+

++

+

--

-

-

-

12 Bu�er_Cone^24 Trehalose11 pH^214 Trehalose^26 pH*NaCl

10 NaCl*Trehalose9 Bu�er_Cone*Trehalose1 pH8 Bu�er_Cone*NaCl3 NaCl

2 Bu�er_Conc7 pH*Trehalose 5 pH*Bu�er_Cone13 NaCl^2

Figure 3. Pareto effects for >0.15 μm (Total)

Pareto E�ects for MT 0.53 Micron

Ter

m

35302520151050

ECHIP

E�ect/1000

++

++

--

-

--

--

--

-

12 Bu�er_Cone^24 Trehalose11 pH^214 Trehalose^26 pH*NaCl

10 NaCl*Trehalose9 Bu�er_Cone*Trehalose1 pH8 Bu�er_Cone*NaCl3 NaCl

2 Bu�er_Conc7 pH*Trehalose5 pH*Bu�er_Cone13 NaCl^2

Figure 4. Pareto effects for >0.53 μm

The next results, shown below present 3-D response

surface plots showing aggregation effects under

different conditions. This empirical view of design

space provides optimization to identify formulations

that minimize aggregation as indicated by higher

particle counts at the two size ranges. Figures 5 and 6

show results for varying pH and stabilizer (trehalose)

while keeping buffer and NaCl concentration constant.

Figure 5. Effect of pH and stabilizer >0.15 μm

x 10

^6

10

5

010

86

24

05

6

7

Bu�er Conc. = 30.0NaCl = 100.0

Trehalose

pH

3

x 10

^4

4

3

2

108

6

24

05

6

7

Bu�er Conc. = 30.0NaCl = 100.0

TrehalosepH

Figure 6. Effect of pH and stabilizer >0.53 μm

Figures 7 and 8 show results for varying NaCl and

stabilizer (trehalose) concentration while keeping

buffer concentration stable and pH varying slightly

from 5.91 to 6.3.

x 10

^6

15

10

5

0

200150

100

500 0

2

4

6

8

10

Bu�er Conc. = 30.0pH = 5.91

NaCl

Treh

alos

e

Figure 7. Effect of NaCl and stabilizer >0.15 μm

x 10

^4

3

2

108

64

20 0

50

100

150

200

Bu�er Conc. = 30.0pH = 6.3

Trehalose

NaC

l

Figure 8. Effect of NaCl and stabilizer >0.53 μm

These, and other results not shown here, were then

compiled to create the minimum particle count

optimization plots shown in Figures 9 and 10.

Tre

hal

ose

5.0 5.5 6.0 6.5 7.0 7.5

0

1

2

3

4

5

6

8

9

7

10 Bu�er Conc. = 30.0NaCl = 71.5

pH=6.30 Trehalose=7.15Value Low Limit High Limit-6.225117e+006 -1.984517e+007 7.394932e+006

2

Total x 10^6

pH

Figure 9. Particle minimization plot >0.15 μmT

reh

alo

se

5.0 5.5 6.0 6.5 7.0 7.5

0

1

2

3

4

5

6

8

9

7

10 Bu�er Conc. = 49.9NaCl = 44.1

pH=6.09 Trehalose=7.82Value Low Limit High Limit-6040.15 -34315.85 22235.54

20

510

20

MT0.53micron x 10^3

pH

Figure 10. Particle minimization plot >0.53 μm

FOR MORE INFORMATION

Please call your Regional Customer Service Center today to learn what Entegris can do for you. Visit entegris.com and select the Contact Us link to find the customer service center nearest you.

TERMS AND CONDITIONS OF SALE

All purchases are subject to Entegris’ Terms and Conditions of Sale. To view and print this information, visit entegris.com and select the Terms & Conditions link in the footer.

www.entegris.com

Entegris®, the Entegris Rings Design®, and other product names are trademarks of Entegris, Inc. as listed on entegris.com/trademarks. All third-party product names, logos, and company names are trademarks or registered trademarks of their respective owners. Use of them does not imply any affiliation, sponsorship, or endorsement by the trademark owner.

©2018-2019 Entegris, Inc. | All rights reserved. | Printed in the USA | 7127-10552TAN-0719

129 Concord RoadBillerica, MA 01821 USA

Tel +1 952 556 4181Fax +1 952 556 8022Toll Free 800 394 4083

Corporate Headquarters Customer Service

CONCLUSIONS—The results shown here indicated that the optimum

formulation chemistries to minimize particle count,

and therefore protein aggregation, were:

pH = 6.1 – 6.3

NaCl = 40 – 70 mM

trehalose = 7 – 8%

Buffer = 50 mM

The AccuSizer can also be used to perform USP <787>

testing at ≥10 and 25 μm, and can be automated using

the A2000 MPA Microplate Analyzer 4.

References1 USP <787>, Subvisible Particulate Matter in Therapeutic Protein

Injuctions, see usp.org

2 NIST reference material 8671, Lot 14HB-D-002, NIST mAb, see

https://www.nist.gov/programs-projects/

nist-monoclonal-antibody-reference-material-8671

3 iformulate, see https://iformulate.net/

4 AccuSizer A2000MPA, see

https://www.youtube.com/watch?v=aMXRwg88 Ro&t=278s